Polynucleotides encoding human ion-exchanger proteins

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

The present application claims the benefit of U.S. ProvisionalApplication No. 60/235,745, which was filed on Sep. 27, 2000 and isherein incorporated by reference in its entirety.

1. INTRODUCTION

The present invention relates to the discovery, identification, andcharacterization of novel human polynucleotides encoding proteins thatshare sequence similarity with mammalian membrane proteins. Theinvention encompasses the described polynucleotides, host cellexpression systems, the encoded proteins, fusion proteins, polypeptidesand peptides, antibodies to the encoded proteins and peptides, andgenetically engineered animals that either lack or over express thedisclosed genes, antagonists and agonists of the proteins, and othercompounds that modulate the expression or activity of the proteinsencoded by the disclosed genes that can be used for diagnosis, drugscreening, clinical trial monitoring, the treatment of diseases anddisorders, and cosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

Membrane proteins can serve as recognition markers, mediate signaltransduction, and can mediate or facilitate the passage of materialsacross the lipid bilayer. As such, membrane proteins, are proven drugtargets.

3. SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification, andcharacterization of nucleotides that encode novel human proteins and thecorresponding amino acid sequences of these proteins. The novel humanproteins (NHPs) described for the first time herein share structuralsimilarity with mammalian sodium-calcium exchanger proteins,sodium-calcium potassium exchanger proteins, and potassium dependentversions of the same.

The novel human nucleic acid sequences described herein encodealternative proteins/open reading frames (ORFs) of 603, 316 and 353amino acids in length (SEQ ID NOS: 2, 4, and 6).

The invention also encompasses agonists and antagonists of the describedNHPs, including small molecules, large molecules, mutant NHPs, orportions thereof, that compete with native NHP, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stemcells (“ES cells”) lines that contain gene trap mutations in a murinehomolog of at least one of the described NHPs. When the unique NHPsequences described in SEQ ID NOS:1-7 are “knocked-out” they provide amethod of identifying phenotypic expression of the particular gene aswell as a method of assigning function to previously unknown genes. Inaddition, animals in which the unique NHP sequences described in SEQ IDNOS:1-7 are “knocked-out” provide a unique source in which to elicitantibodies to homologous and orthologous proteins that would have beenpreviously viewed by the immune system as “self” and therefore wouldhave failed to elicit significant antibody responses. To these ends,gene trapped knockout ES cells have been generated in murine homologs ofthe described NHPs.

Additionally, the unique NHP sequences described in SEQ ID NOS:1-7 areuseful for the identification of protein coding sequence and mapping aunique gene to a particular chromosome. These sequences identify actual,biologically relevant, exon splice junctions as opposed to those thatmight have been predicted bioinformatically from genomic sequence alone.The sequences of the present invention are also useful as additional DNAmarkers for restriction fragment length polymorphism (RFLP) analysis,and in forensic biology.

Further, the present invention also relates to processes for identifyingcompounds that modulate, i.e., act as agonists or antagonists, of NHPexpression and/or NHP activity that utilize purified preparations of thedescribed NHPs and/or NHP product, or cells expressing the same. Suchcompounds can be used as therapeutic agents for the treatment of any ofa wide variety of symptoms associated with biological disorders orimbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

The Sequence Listing provides the sequences of the NHP ORFs encoding thedescribed NHP amino acid sequences. SEQ ID NO:7 describes apolynucleotide encoding a NHP ORF with regions of flanking sequence.

5. DETAILED DESCRIPTION OF THE INVENTION

The NHPs described for the first time herein are novel proteins that maybe expressed in, inter alia, human cell lines, fetal brain, brain,pituitary, cerebellum, spinal cord, thymus, spleen, bone marrow, lymphnode, trachea, lung, kidney, fetal liver, liver, prostate, testis,thyroid, adrenal gland, pancreas, salivary gland, stomach, smallintestine, colon, skeletal muscle, heart, uterus, placenta, mammarygland, adipose, skin, esophagus, bladder, cervix, rectum, pericardium,hypothalamus, ovary, fetal kidney, fetal lung, gall bladder, tongue, 6-,9-, and 12 week embryo, adenocarcinoma, and embryonic carcinoma cells.

The present invention encompasses the nucleotides presented in theSequence Listing, host cells expressing such nucleotides, the expressionproducts of such nucleotides, and: (a) nucleotides that encode mammalianhomologs of the described genes, including the specifically describedNHPs, and the NHP products; (b) nucleotides that encode one or moreportions of the NHPs that correspond to functional domains, and thepolypeptide products specified by such nucleotide sequences, includingbut not limited to the novel regions of any active domain(s); (c)isolated nucleotides that encode mutant versions, engineered ornaturally occurring, of the described NHPs in which all or a part of atleast one domain is deleted or altered, and the polypeptide productsspecified by such nucleotide sequences, including but not limited tosoluble proteins and peptides in which all or a portion of the signal(or hydrophobic transmembrane) sequence is deleted; (d) nucleotides thatencode chimeric fusion proteins containing all or a portion of a codingregion of an NHP, or one of its domains (e.g., a receptor or ligandbinding domain, accessory protein/self-association domain, etc.) fusedto another peptide or polypeptide; or (e) therapeutic or diagnosticderivatives of the described polynucleotides such as oligonucleotides,antisense polynucleotides, ribozymes, dsRNA, or gene therapy constructscomprising a sequence first disclosed in the Sequence Listing.

As discussed above, the present invention includes: (a) the human DNAsequences presented in the Sequence Listing (and vectors comprising thesame) and additionally contemplates any nucleotide sequence encoding acontiguous NHP open reading frame (ORF) that hybridizes to a complementof a DNA sequence presented in the Sequence Listing under highlystringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3)and encodes a functionally equivalent expression product. Additionallycontemplated are any nucleotide sequences that hybridize to thecomplement of a DNA sequence that encodes and expresses an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989, supra), yet still encodes a functionally equivalent NHP product.Functional equivalents of a NHP include naturally occurring NHPs presentin other species and mutant NHPs whether naturally occurring orengineered (by site directed mutagenesis, gene shuffling, directedevolution as described in, for example, U.S. Pat. No. 5,837,458). Theinvention also includes degenerate nucleic acid variants of thedisclosed NHP polynucleotide sequences.

Additionally contemplated are polynucleotides encoding NHP ORFs, ortheir functional equivalents, encoded by polynucleotide sequences thatare about 99, 95, 90, or about 85 percent similar or identical tocorresponding regions of the nucleotide sequences of the SequenceListing (as measured by BLAST sequence comparison analysis using, forexample, the GCG sequence analysis package using standard defaultsettings).

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, thedescribed NHP gene nucleotide sequences. Such hybridization conditionsmay be highly stringent or less highly stringent, as described above. Ininstances where the nucleic acid molecules are deoxyoligonucleotides(“DNA oligos”), such molecules are generally about 16 to about 100 baseslong, or about 20 to about 80, or about 34 to about 45 bases long, orany variation or combination of sizes represented therein thatincorporate a contiguous region of sequence first disclosed in theSequence Listing. Such oligonucleotides can be used in conjunction withthe polymerase chain reaction (PCR) to screen libraries, isolate clones,and prepare cloning and sequencing templates, etc.

Alternatively, such NHP oligonucleotides can be used as hybridizationprobes for screening libraries, and assessing gene expression patterns(particularly using a micro array or high-throughput “chip” format).Additionally, a series of the described NHP oligonucleotide sequences,or the complements thereof, can be used to represent all or a portion ofthe described NHP sequences. An oligonucleotide or polynucleotidesequence first disclosed in at least a portion of one or more of thesequences of SEQ ID NOS: 1-7 can be used as a hybridization probe inconjunction with a solid support matrix/substrate (resins, beads,membranes, plastics, polymers, metal or metallized substrates,crystalline or polycrystalline substrates, etc.). Of particular note arespatially addressable arrays (i.e., gene chips, microtiter plates, etc.)of oligonucleotides and polynucleotides, or corresponding oligopeptidesand polypeptides, wherein at least one of the biopolymers present on thespatially addressable array comprises an oligonucleotide orpolynucleotide sequence first disclosed in at least one of the sequencesof SEQ ID NOS: 1-7, or an amino acid sequence encoded thereby. Methodsfor attaching biopolymers to, or synthesizing biopolymers on, solidsupport matrices, and conducting binding studies thereon are disclosedin, inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752, 5,744,305,4,631,211, 5,445,934, 5,252,743, 4,713,326, 5,424,186, and 4,689,405,the disclosures of which are herein incorporated by reference in theirentirety.

Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-7 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is within a range of between about 8 to about2000 nucleotides. Preferably the probes consist of 60 nucleotides andmore preferably 25 nucleotides from the sequences first disclosed in SEQID NOS:1-7.

For example, a series of the described oligonucleotide sequences, or thecomplements thereof, can be used in chip format to represent all or aportion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length can partially overlap each other and/or thesequence may be represented using oligonucleotides that do not overlap.Accordingly, the described polynucleotide sequences shall typicallycomprise at least about two or three distinct oligonucleotide sequencesof at least about 8 nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences canbegin at any nucleotide present within a sequence in the SequenceListing and proceed in either a sense (5′-to-3′) orientation vis-a-visthe described sequence or in an antisense orientation.

Microarray-based analysis allows the discovery of broad patterns ofgenetic activity, providing new understanding of gene functions andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-7 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel componentsor gene functions that manifest themselves as novel phenotypes.

Probes consisting of sequences first disclosed in SEQ ID NOS:1-7 canalso be used in the identification, selection and validation of novelmolecular targets for drug discovery. The use of these unique sequencespermits the direct confirmation of drug targets and recognition of drugdependent changes in gene expression that are modulated through pathwaysdistinct from the drugs intended target. These unique sequencestherefore also have utility in defining and monitoring both drug actionand toxicity.

As an example of utility, the sequences first disclosed in SEQ IDNOS:1-7 can be utilized in microarrays or other assay formats, to screencollections of genetic material from patients who have a particularmedical condition. These investigations can also be carried out usingthe sequences first disclosed in SEQ ID NOS:1-7 in silico and bycomparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

Thus the sequences first disclosed in SEQ ID NOS:1-7 can be used toidentify mutations associated with a particular disease and also as adiagnostic or prognostic assay.

Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence in conjunctionwith the presence of one or more specific oligonucleotide sequence(s)first disclosed in the SEQ ID NOS: 1-7. Alternatively, a restriction mapspecifying the relative positions of restriction endonuclease digestionsites, or various palindromic or other specific oligonucleotidesequences can be used to structurally describe a given sequence. Suchrestriction maps, which are typically generated by widely availablecomputer programs (e.g., the University of Wisconsin GCG sequenceanalysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor, Mich.,etc.), can optionally be used in conjunction with one or more discretenucleotide sequence(s) present in the sequence that can be described bythe relative position of the sequence relative to one or more additionalsequence(s) or one or more restriction sites present in the disclosedsequence. For oligonucleotide probes, highly stringent conditions mayrefer, e.g., to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). These nucleic acid moleculesmay encode or act as NHP gene antisense molecules, useful, for example,in NHP gene regulation (for and/or as antisense primers in amplificationreactions of NHP gene nucleic acid sequences). With respect to NHP generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences may be used as part of ribozymeand/or triple helix sequences that are also useful for NHP generegulation.

Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety that is selectedfrom the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide can also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide will compriseat least one modified phosphate backbone selected from the groupconsisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

Oligonucleotides of the invention can be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides can be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

Low stringency conditions are well known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y.

Alternatively, suitably labeled NHP nucleotide probes can be used toscreen a human genomic library using appropriately stringent conditionsor by PCR. The identification and characterization of human genomicclones is helpful for identifying polymorphisms (including, but notlimited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

For example, the present sequences can be used in restriction fragmentlength polymorphism (RFLP) analysis to identify specific individuals. Inthis technique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification (as generally described in U.S. Pat. No. 5,272,057,incorporated herein by reference). In addition, the sequences of thepresent invention can be used to provide polynucleotide reagents, e.g.,PCR primers, targeted to specific loci in the human genome, which canenhance the reliability of DNA-based forensic identifications by, forexample, providing another “identification marker” (i.e., another DNAsequence that is unique to a particular individual). Actual basesequence information can be used for identification as an accuratealternative to patterns formed by restriction enzyme generatedfragments.

Further, a NHP gene homolog can be isolated from nucleic acid from anorganism of interest by performing PCR using two degenerate or “wobble”oligonucleotide primer pools designed on the basis of amino acidsequences within the NHP products disclosed herein. The template for thereaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from human or non-human cell lines ortissue known or suspected to express an allele of a NHP gene. The PCRproduct can be subcloned and sequenced to ensure that the amplifiedsequences represent the sequence of the desired NHP gene. The PCRfragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

PCR technology can also be used to isolate full length cDNA sequences.For example, RNA can be isolated, following standard procedures, from anappropriate cellular or tissue source (i.e., one known, or suspected, toexpress a NHP gene). A reverse transcription (RT) reaction can beperformed on the RNA using an oligonucleotide primer specific for themost 5′ end of the amplified fragment for the priming of first strandsynthesis. The resulting RNA/DNA hybrid may then be “tailed” using astandard terminal transferase reaction, the hybrid may be digested withRNase H, and second strand synthesis may then be primed with acomplementary primer. Thus, cDNA sequences upstream of the amplifiedfragment can be isolated. For a review of cloning strategies that can beused, see e.g., Sambrook et al., 1989, supra.

A cDNA encoding a mutant NHP sequence can be isolated, for example, byusing PCR. In this case, the first cDNA strand may be synthesized byhybridizing an oligo-dT oligonucleotide to mRNA isolated from tissueknown or suspected to be expressed in an individual putatively carryinga mutant NHP allele, and by extending the new strand with reversetranscriptase. The second strand of the cDNA is then synthesized usingan oligonucleotide that hybridizes specifically to the 5′ end of thenormal sequence. Using these two primers, the product is then amplifiedvia PCR, optionally cloned into a suitable vector, and subjected to DNAsequence analysis through methods well known to those of skill in theart. By comparing the DNA sequence of the mutant NHP allele to that of acorresponding normal NHP allele, the mutation(s) responsible for theloss or alteration of function of the mutant NHP gene product can beascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of or known to carry a mutant NHP allele(e.g., a person manifesting a NHP-associated phenotype such as, forexample, osteoporosis, obesity, high blood pressure, connective tissuedisorders, infertility, etc.), or a cDNA library can be constructedusing RNA from a tissue known, or suspected, to express a mutant NHPallele. A normal NHP gene, or any suitable fragment thereof, can then belabeled and used as a probe to identify the corresponding mutant NHPallele in such libraries. Clones containing mutant NHP sequences canthen be purified and subjected to sequence analysis according to methodswell known to those skilled in the art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant NHP allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue can be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against a normal NHP product, as described below. (For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold SpringHarbor.) Additionally, screening can be accomplished by screening withlabeled NHP fusion proteins, such as, for example, alkalinephosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In caseswhere a NHP mutation results in an expression product with alteredfunction (e.g., as a result of a missense or a frameshift mutation),polyclonal antibodies to NHP are likely to cross-react with acorresponding mutant NHP expression product. Library clones detected viatheir reaction with such labeled antibodies can be purified andsubjected to sequence analysis according to methods well known in theart.

The invention also encompasses (a) DNA vectors that contain any of theforegoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

The present invention also encompasses antibodies and anti-idiotypicantibodies (including Fab fragments), antagonists and agonists of a NHP,as well as compounds or nucleotide constructs that inhibit expression ofa NHP sequence (transcription factor inhibitors, antisense and ribozymemolecules, or open reading frame sequence or regulatory sequencereplacement constructs), or promote the expression of a NHP (e.g.,expression constructs in which NHP coding sequences are operativelyassociated with expression control elements such as promoters,promoter/enhancers, etc.).

The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotide sequences,antibodies, antagonists and agonists can be useful for the detection ofmutant NHPs or inappropriately expressed NHPs for the diagnosis ofdisease. The NHP proteins or peptides, NHP fusion proteins, NHPnucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of NHP inthe body. The use of engineered host cells and/or animals may offer anadvantage in that such systems allow not only for the identification ofcompounds that bind to the endogenous receptor for an NHP, but can alsoidentify compounds that trigger NHP-mediated activities or pathways.

Finally, the NHP products can be used as therapeutics. For example,soluble derivatives such as NHP peptides/domains corresponding to NHPs,NHP fusion protein products (especially NHP-Ig fusion proteins, i.e.,fusions of a NHP, or a domain of a NHP, to an IgFc), NHP antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists oragonists (including compounds that modulate or act on downstream targetsin a NHP-mediated pathway) can be used to directly treat diseases ordisorders. For instance, the administration of an effective amount ofsoluble NHP, or a NHP-IgFc fusion protein or an anti-idiotypic antibody(or its Fab) that mimics the NHP could activate or effectivelyantagonize the endogenous NHP receptor. Nucleotide constructs encodingsuch NHP products can be used to genetically engineer host cells toexpress such products in vivo; these genetically engineered cellsfunction as “bioreactors” in the body delivering a continuous supply ofa NHP, a NHP peptide, or a NHP fusion protein to the body. Nucleotideconstructs encoding functional NHPs, mutant NHPs, as well as antisenseand ribozyme molecules can also be used in “gene therapy” approaches forthe modulation of NHP expression. Thus, the invention also encompassespharmaceutical formulations and methods for treating biologicaldisorders.

Various aspects of the invention are described in greater detail in thesubsections below.

5.1 The NHP Sequences

The cDNA sequences and the corresponding deduced amino acid sequences ofthe described NHPs are presented in the Sequence Listing. The NHPnucleotides were obtained from clustered genomic sequence, ESTs, andcDNAs from brain and pituitary gland cDNA libraries (Edge Biosystems,Gaithersburg, Md.). The gene encoding the described NHPs is apparentlyencoded on human chromosome 14 (see GENBANK accession no. AL118559).

A number of polymorphism were identified during the sequencing of theNHPs including a C/G at the nucleotide position represented by, forexample, position 1147 of SEQ ID NO: 1 (which can result in a pro or alaat corresponding amino acid (aa) position 383), a T/G at nucleotideposition 1163 (which can result in an val or gly at aa position 388),and a T/G at position 1193 (which can result in a val or gly at aaposition 398). The present invention contemplates sequences comprisingany of the above polymorphisms, as well as any and all combinations andpermutations of the above.

An additional application of the described novel human polynucleotidesequences is their use in the molecular mutagenesis/evolution ofproteins that are at least partially encoded by the described novelsequences using, for example, polynucleotide shuffling or relatedmethodologies. Such approaches are described in U.S. Pat. Nos. 5,830,721and 5,837,458, which are herein incorporated by reference in theirentirety.

NHP gene products can also be expressed in transgenic animals. Animalsof any species, including, but not limited to, worms, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, birds, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees may be used togenerate NHP transgenic animals.

Any technique known in the art may be used to introduce a NHP transgeneinto animals to produce the founder lines of transgenic animals. Suchtechniques include, but are not limited to pronuclear microinjection(Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191);retrovirus mediated gene transfer into germ lines (Van der Putten etal., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting inembryonic stem cells (Thompson et al., 1989, Cell 56:313-321);electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); andsperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723);etc. For a review of such techniques, see Gordon, 1989, TransgenicAnimals, Intl. Rev. Cytol. 115:171-229, which is incorporated byreference herein in its entirety.

The present invention provides for transgenic animals that carry the NHPtransgene in all their cells, as well as animals that carry thetransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. The transgene may be integrated as asingle transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art.

When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

The transgene can also be selectively introduced into a particular celltype, thus inactivating the endogenous NHP gene in only that cell type,by following, for example, the teaching of Gu et al., 1994, Science,265:103-106. The regulatory sequences required for such a cell-typespecific inactivation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant NHP gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques that include but are not limited to Northern blot analysis oftissue samples obtained from the animal, in situ hybridization analysis,and RT-PCR. Samples of NHP gene-expressing tissue, may also be evaluatedimmunocytochemically using antibodies specific for the NHP transgeneproduct.

5.2 NHPS and NHP Polypeptides

NHPs, NHP polypeptides, NHP peptide fragments, mutated, truncated, ordeleted forms of the NHPs, and/or NHP fusion proteins can be preparedfor a variety of uses. These uses include, but are not limited to, thegeneration of antibodies, as reagents in diagnostic assays, for theidentification of other cellular gene products related to a NHP, asreagents in assays for screening for compounds that can be used aspharmaceutical reagents useful in the therapeutic treatment of mental,biological, or medical disorders and disease. Given the similarityinformation and expression data, the described NHPs can be targeted (bydrugs, oligos, antibodies, etc.,) in order to treat disease, or totherapeutically augment the efficacy of, for example, chemotherapeuticagents used in the treatment of cancer, arthritis, or as antiviralagents.

The Sequence Listing discloses the amino acid sequences encoded by thedescribed NHP sequences. The NHPs display initiator methionines in DNAsequence contexts consistent with translation initiation sites, and ahydrophobic region near the N-terminus that may serve as a signalsequence, which indicates that the described NHPs can be secreted,membrane-associated, or cytoplasmic.

The NHP amino acid sequences of the invention include the amino acidsequence presented in the Sequence Listing as well as analogues andderivatives thereof. Further, corresponding NHP homologues from otherspecies are encompassed by the invention. In fact, any NHP proteinencoded by the NHP nucleotide sequences described above are within thescope of the invention as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code is wellknown, and, accordingly, each amino acid presented in the SequenceListing, is generically representative of the well known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al. eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference) are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

The invention also encompasses proteins that are functionally equivalentto the NHPs encoded by the presently described nucleotide sequences asjudged by any of a number of criteria, including, but not limited to,the ability to bind and cleave a substrate of a NHP, or the ability toeffect an identical or complementary downstream pathway, or a change incellular metabolism (e.g., proteolytic activity, ion flux, tyrosinephosphorylation, etc.). Such functionally equivalent NHP proteinsinclude, but are not limited to, additions or substitutions of aminoacid residues within the amino acid sequence encoded by the NHPnucleotide sequences described above, but that result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

A variety of host-expression vector systems can be used to express theNHP nucleotide sequences of the invention. Where, as in the presentinstance, the NHP peptide or polypeptide is thought to be membraneprotein, the hydrophobic regions of the protein can be excised and theresulting soluble peptide or polypeptide can be recovered from theculture media. Such expression systems also encompass engineered hostcells that express a NHP, or functional equivalent, in situ.Purification or enrichment of a NHP from such expression systems can beaccomplished using appropriate detergents and lipid micelles and methodswell known to those skilled in the art. However, such engineered hostcells themselves may be used in situations where it is important notonly to retain the structural and functional characteristics of the NHP,but to assess biological activity, e.g., in drug screening assays.

The expression systems that may be used for purposes of the inventioninclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing NHP nucleotidesequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing NHP nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing NHP sequences; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing NHP nucleotide sequences; or mammalian cell systems(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited, to the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouye& Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Pharmacia or American Type Culture Collection) can also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The PGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target expression productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign polynucleotide sequences.The virus grows in Spodoptera frugiperda cells. A NHP coding sequencecan be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion ofNHP coding sequence will result in inactivation of the polyhedrin geneand production of non-occluded recombinant virus (i.e., virus lackingthe proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted sequence is expressed (e.g., see Smith et al.,1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the NHP nucleotide sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric sequence may thenbe inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing a NHP product in infected hosts (e.g.,See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659).Specific initiation signals may also be required for efficienttranslation of inserted NHP nucleotide sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire NHP gene or cDNA, including its own initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only a portion of a NHP coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, must be provided. Furthermore, the initiation codonmust be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (See Bitter et al., 1987,Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the correct modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells that possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theNHP sequences described above can be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci, which in turn canbe cloned and expanded into cell lines. This method may advantageouslybe used to engineer cell lines that express the NHP product. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of the NHPproduct.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes, whichcan be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

Alternatively, any fusion protein can be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the sequence of interest is subclonedinto a vaccinia recombination plasmid such that the sequence's openreading frame is translationally fused to an amino-terminal tagconsisting of six histidine residues. Extracts from cells infected withrecombinant vaccinia virus are loaded onto Ni²⁺.nitriloaceticacid-agarose columns and histidine-tagged proteins are selectivelyeluted with imidazole-containing buffers.

Also encompassed by the present invention are fusion proteins thatdirect the NHP to a target organ and/or facilitate transport across themembrane into the cytosol. Conjugation of NHPs to antibody molecules ortheir Fab fragments could be used to target cells bearing a particularepitope. Attaching the appropriate signal sequence to the NHP would alsotransport the NHP to the desired location within the cell. Alternativelytargeting of NHP or its nucleic acid sequence might be achieved usingliposome or lipid complex based delivery systems. Such technologies aredescribed in “Liposomes: A Practical Approach”, New, R.R.C., ed., OxfordUniversity Press, New York and in U.S. Pat. Nos. 4,594,595, 5,459,127,5,948,767 and 6,110,490 and their respective disclosures, which areherein incorporated by reference in their entirety. Additionallyembodied are novel protein constructs engineered in such a way that theyfacilitate transport of the NHP to the target site or desired organ,where they cross the cell membrane and/or the nucleus where the NHP canexert its functional activity. This goal may be achieved by coupling ofthe NHP to a cytokine or other ligand that provides targetingspecificity, and/or to a protein transducing domain (see generally U.S.applications Ser. Nos. 60/111,701 and 60/056,713, both of which areherein incorporated by reference, for examples of such transducingsequences) to facilitate passage across cellular membranes and canoptionally be engineered to include nuclear localization.

5.3 Antibodies to NHP Products

Antibodies that specifically recognize one or more epitopes of a NHP, orepitopes of conserved variants of a NHP, or peptide fragments of a NHPare also encompassed by the invention. Such antibodies include but arenot limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention may be used, for example, in thedetection of NHP in a biological sample and may, therefore, be utilizedas part of a diagnostic or prognostic technique whereby patients may betested for abnormal amounts of NHP. Such antibodies may also be utilizedin conjunction with, for example, compound screening schemes for theevaluation of the effect of test compounds on expression and/or activityof a NHP expression product. Additionally, such antibodies can be usedin conjunction gene therapy to, for example, evaluate the normal and/orengineered NHP-expressing cells prior to their introduction into thepatient. Such antibodies may additionally be used as a method for theinhibition of abnormal NHP activity. Thus, such antibodies may,therefore, be utilized as part of treatment methods.

For the production of antibodies, various host animals may be immunizedby injection with a NHP, an NHP peptide (e.g., one corresponding to afunctional domain of an NHP), truncated NHP polypeptides (NHP in whichone or more domains have been deleted), functional equivalents of theNHP or mutated variant of the NHP. Such host animals may include but arenot limited to pigs, rabbits, mice, goats, and rats, to name but a few.Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund'sadjuvant (complete and incomplete), mineral salts such as aluminumhydroxide or aluminum phosphate, chitosan, surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, and potentially useful human adjuvants such as BCG (bacilleCalmette-Guerin) and Corynebacterium parvum. Alternatively, the immuneresponse could be enhanced by combination and or coupling with moleculessuch as keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid,ovalbumin, cholera toxin or fragments thereof. Polyclonal antibodies areheterogeneous populations of antibody molecules derived from the sera ofthe immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 6,075,181 and5,877,397 and their respective disclosures, which are hereinincorporated by reference in their entirety. Also encompassed by thepresent invention is the use of fully humanized monoclonal antibodies asdescribed in U.S. Pat. No. 6,150,584 and respective disclosures, whichare herein incorporated by reference in their entirety.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 341:544-546) can be adapted to produce single chainantibodies against NHP expression products. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include, but are notlimited to: the F(ab′)₂ fragments, which can be produced by pepsindigestion of the antibody molecule and the Fab fragments, which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to a NHP can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” a given NHP, using techniques well known tothose skilled in the art. (See, e.g., Greenspan & Bona, 1993, FASEB J7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438). Forexample antibodies that bind to a NHP domain and competitively inhibitthe binding of NHP to its cognate receptor can be used to generateanti-idiotypes that “mimic” the NHP and, therefore, bind and activate orneutralize a receptor. Such anti-idiotypic antibodies or Fab fragmentsof such anti-idiotypes can be used in therapeutic regimens involving aNHP mediated pathway.

Additionally given the high degree of relatedness of mammalian NHPs, thepresently described knock-out mice (having never seen NHP, and thusnever been tolerized to NHP) have a unique utility, as they can beadvantageously applied to the generation of antibodies against thedisclosed mammalian NHP (i.e., NHP will be immunogenic in NHP knock-outanimals).

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 7 <210> SEQ ID NO 1 <211> LENGTH: 1812<212> TYPE: DNA <213> ORGANISM: homo sapiens <400> SEQUENCE: 1atggcgctcc gcgggaccct ccggccgctc aaagttcgca ggaggcgaga ga#tgctgccg     60cagcaagtcg gcttcgtgtg cgcggtgctg gccctggtgt gctgtgcgtc cg#gcctcttc    120ggcagcttgg ggcacaaaac agcttctgct agcaaacgtg tcctgccaga ca#catggaga    180aatagaaagt tgatggcccc agtgaatggg acacagacag ccaagaactg ca#cagatcct    240gcgattcacg agttccccac agatctgttc tccaataagg agcgacagca cg#gagccgtc    300ctgctgcaca tccttggtgc tctgtatatg ttctatgcct tggccatagt gt#gcgatgac    360ttctttgttc cgtctctaga gaagatctgt gagagactcc atctgagcga ag#atgtggct    420ggagccacct tcatggctgc aggaagctca acgccagagc tgtttgcgtc tg#ttattggg    480gtgttcatca cccaygggga cgtcggggtg ggcaccatcg tgggctctgc tg#tgttcaac    540atcctgtgca taattggagt gtgcggactg tttgctggcc aggtggtccg tc#tgacgtgg    600tgggccgtgt gccgagactc cgtgtactac accatctctg tcatcgtgct ca#tcgtgttc    660atatatgatg aacaaattgt gtggtgggaa ggcctggtgc tcatcatctt gt#atgtgttt    720tatattctga tcatgaagta caatgtgaag atgcaagcct ttttcacagt ca#aacaaaag    780agcattgcaa acggtaaccc ggtcaacagt gagctggagg ctgtgaagga ga#agccacag    840tatggcaaga accccgtggt gatggtggac gagattatga gctccagccc tc#ccaagttc    900accttccctg aagcaggctt acgaatcatg atcaccaata agtttggacc ca#ggacccga    960ctacggatgg ccagcaggat catcattaat gagcggcaga gactgatcaa ct#cggccaat   1020ggtgtgagca gtaagccgct tcaaaacggg aggcacgaga acattgagaa cg#ggaatgtt   1080cctgtggaaa accccgaaga ccctcagcag aatcaggagc agcagccgcc gc#cacagcca   1140ccaccgccag agccagagcc ggtggaggct gacttcctgt cccccttctc cg#tgccggag   1200gccagagggg acaaggtcaa gtgggtgttc acctggcccc tcatcttcct cc#tgtgcgtc   1260accattccca actgcagcaa gccccgctgg gagaagttct tcatggtcac ct#tcatcacc   1320gccacgctgt ggatcgctgt gttctcctac atcatggtgt ggctggtgac ta#ttatcgga   1380tacacacttg ggatcccgga tgtcatcatg ggcattactt tcctggcagc ag#ggacaagt   1440gttccagact gcatggccag cctaattgtg gcgagacaag gccttgggga ca#tggcagtc   1500tccaacacca taggaagcaa cgtgtttgac atcctggtag gacttggtgt ac#cgtggggc   1560ctgcagacca tggttgttaa ttatggatca acagtgaaga tcaacagccg gg#ggctggtc   1620tattccgtgg tcctgttgct gggctctgtc gctctcaccg tcctcggcat cc#acctaaac   1680aagtggcgac tggaccggaa gctgggtgtc tacgtgctgg ttctctacgc ca#tcttcttg   1740tgcttctcca taatgataga gtttaacgtc tttaccttcg tcaacttgcc ga#tgtgccgg   1800 gaagacgatt ag               #                  #                   #     1812 <210> SEQ ID NO 2 <211> LENGTH: 603<212> TYPE: PRT <213> ORGANISM: homo sapiens <400> SEQUENCE: 2Met Ala Leu Arg Gly Thr Leu Arg Pro Leu Ly #s Val Arg Arg Arg Arg 1               5   #                10   #                15Glu Met Leu Pro Gln Gln Val Gly Phe Val Cy #s Ala Val Leu Ala Leu            20       #            25       #            30Val Cys Cys Ala Ser Gly Leu Phe Gly Ser Le #u Gly His Lys Thr Ala        35           #        40           #        45Ser Ala Ser Lys Arg Val Leu Pro Asp Thr Tr #p Arg Asn Arg Lys Leu    50               #    55               #    60Met Ala Pro Val Asn Gly Thr Gln Thr Ala Ly #s Asn Cys Thr Asp Pro65                   #70                   #75                   #80Ala Ile His Glu Phe Pro Thr Asp Leu Phe Se #r Asn Lys Glu Arg Gln                85   #                90   #                95His Gly Ala Val Leu Leu His Ile Leu Gly Al #a Leu Tyr Met Phe Tyr            100       #           105       #           110Ala Leu Ala Ile Val Cys Asp Asp Phe Phe Va #l Pro Ser Leu Glu Lys        115           #       120           #       125Ile Cys Glu Arg Leu His Leu Ser Glu Asp Va #l Ala Gly Ala Thr Phe    130               #   135               #   140Met Ala Ala Gly Ser Ser Thr Pro Glu Leu Ph #e Ala Ser Val Ile Gly145                 1 #50                 1 #55                 1 #60Val Phe Ile Thr His Gly Asp Val Gly Val Gl #y Thr Ile Val Gly Ser                165   #               170   #               175Ala Val Phe Asn Ile Leu Cys Ile Ile Gly Va #l Cys Gly Leu Phe Ala            180       #           185       #           190Gly Gln Val Val Arg Leu Thr Trp Trp Ala Va #l Cys Arg Asp Ser Val        195           #       200           #       205Tyr Tyr Thr Ile Ser Val Ile Val Leu Ile Va #l Phe Ile Tyr Asp Glu    210               #   215               #   220Gln Ile Val Trp Trp Glu Gly Leu Val Leu Il #e Ile Leu Tyr Val Phe225                 2 #30                 2 #35                 2 #40Tyr Ile Leu Ile Met Lys Tyr Asn Val Lys Me #t Gln Ala Phe Phe Thr                245   #               250   #               255Val Lys Gln Lys Ser Ile Ala Asn Gly Asn Pr #o Val Asn Ser Glu Leu            260       #           265       #           270Glu Ala Val Lys Glu Lys Pro Gln Tyr Gly Ly #s Asn Pro Val Val Met        275           #       280           #       285Val Asp Glu Ile Met Ser Ser Ser Pro Pro Ly #s Phe Thr Phe Pro Glu    290               #   295               #   300Ala Gly Leu Arg Ile Met Ile Thr Asn Lys Ph #e Gly Pro Arg Thr Arg305                 3 #10                 3 #15                 3 #20Leu Arg Met Ala Ser Arg Ile Ile Ile Asn Gl #u Arg Gln Arg Leu Ile                325   #               330   #               335Asn Ser Ala Asn Gly Val Ser Ser Lys Pro Le #u Gln Asn Gly Arg His            340       #           345       #           350Glu Asn Ile Glu Asn Gly Asn Val Pro Val Gl #u Asn Pro Glu Asp Pro        355           #       360           #       365Gln Gln Asn Gln Glu Gln Gln Pro Pro Pro Gl #n Pro Pro Pro Pro Glu    370               #   375               #   380Pro Glu Pro Val Glu Ala Asp Phe Leu Ser Pr #o Phe Ser Val Pro Glu385                 3 #90                 3 #95                 4 #00Ala Arg Gly Asp Lys Val Lys Trp Val Phe Th #r Trp Pro Leu Ile Phe                405   #               410   #               415Leu Leu Cys Val Thr Ile Pro Asn Cys Ser Ly #s Pro Arg Trp Glu Lys            420       #           425       #           430Phe Phe Met Val Thr Phe Ile Thr Ala Thr Le #u Trp Ile Ala Val Phe        435           #       440           #       445Ser Tyr Ile Met Val Trp Leu Val Thr Ile Il #e Gly Tyr Thr Leu Gly    450               #   455               #   460Ile Pro Asp Val Ile Met Gly Ile Thr Phe Le #u Ala Ala Gly Thr Ser465                 4 #70                 4 #75                 4 #80Val Pro Asp Cys Met Ala Ser Leu Ile Val Al #a Arg Gln Gly Leu Gly                485   #               490   #               495Asp Met Ala Val Ser Asn Thr Ile Gly Ser As #n Val Phe Asp Ile Leu            500       #           505       #           510Val Gly Leu Gly Val Pro Trp Gly Leu Gln Th #r Met Val Val Asn Tyr        515           #       520           #       525Gly Ser Thr Val Lys Ile Asn Ser Arg Gly Le #u Val Tyr Ser Val Val    530               #   535               #   540Leu Leu Leu Gly Ser Val Ala Leu Thr Val Le #u Gly Ile His Leu Asn545                 5 #50                 5 #55                 5 #60Lys Trp Arg Leu Asp Arg Lys Leu Gly Val Ty #r Val Leu Val Leu Tyr                565   #               570   #               575Ala Ile Phe Leu Cys Phe Ser Ile Met Ile Gl #u Phe Asn Val Phe Thr            580       #           585       #           590Phe Val Asn Leu Pro Met Cys Arg Glu Asp As #p         595          #       600 <210> SEQ ID NO 3 <211> LENGTH: 951 <212> TYPE: DNA<213> ORGANISM: homo sapiens <400> SEQUENCE: 3atggtggacg agattatgag ctccagccct cccaagttca ccttccctga ag#caggctta     60cgaatcatga tcaccaataa gtttggacct aggacccgac tacggatggc ca#gcaggatc    120atcattaatg agcggcagag actgatcaac tcggccaatg gtgtgagcag ta#agccgctt    180caaaacggga ggcacgagaa cattgagaac gggaatgttc ctgtggaaaa cc#ccgaagac    240cctcagcaga atcaggagca gcagccgccg ccacagccac caccgccaga gc#cagagccg    300gtggaggctg acttcctgtc ccccttctcc gtgccggagg ccagagggga ca#aggtcaag    360tgggtgttca cctggcccct catcttcctc ctgtgcgtca ccattcccaa ct#gcagcaag    420ccccgctggg agaagttctt catggtcacc ttcatcaccg ccacgctgtg ga#tcgctgtg    480ttctcctaca tcatggtgtg gctggtgact attatcggat acacacttgg ga#tcccggat    540gtcatcatgg gcattacttt cctggcagca gggacaagtg ttccagactg ca#tggccagc    600ctaattgtgg cgagacaagg ccttggggac atggcagtct ccaacaccat ag#gaagcaac    660gtgtttgaca tcctggtagg acttggtgta ccgtggggcc tgcagaccat gg#ttgttaat    720tatggatcaa cagtgaagat caacagccgg gggctggtct attccgtggt cc#tgttgctg    780ggctctgtcg ctctcaccgt cctcggcatc cacctaaaca agtggcgact gg#accggaag    840ctgggtgtct acgtgctggt tctctacgcc atcttcttgt gcttctccat aa#tgatagag    900tttaacgtct ttaccttcgt caacttgccg atgtgccggg aagacgatta g #            951 <210> SEQ ID NO 4 <211> LENGTH: 316 <212> TYPE: PRT<213> ORGANISM: homo sapiens <400> SEQUENCE: 4Met Val Asp Glu Ile Met Ser Ser Ser Pro Pr #o Lys Phe Thr Phe Pro 1               5   #                10   #                15Glu Ala Gly Leu Arg Ile Met Ile Thr Asn Ly #s Phe Gly Pro Arg Thr            20       #            25       #            30Arg Leu Arg Met Ala Ser Arg Ile Ile Ile As #n Glu Arg Gln Arg Leu        35           #        40           #        45Ile Asn Ser Ala Asn Gly Val Ser Ser Lys Pr #o Leu Gln Asn Gly Arg    50               #    55               #    60His Glu Asn Ile Glu Asn Gly Asn Val Pro Va #l Glu Asn Pro Glu Asp65                   #70                   #75                   #80Pro Gln Gln Asn Gln Glu Gln Gln Pro Pro Pr #o Gln Pro Pro Pro Pro                85   #                90   #                95Glu Pro Glu Pro Val Glu Ala Asp Phe Leu Se #r Pro Phe Ser Val Pro            100       #           105       #           110Glu Ala Arg Gly Asp Lys Val Lys Trp Val Ph #e Thr Trp Pro Leu Ile        115           #       120           #       125Phe Leu Leu Cys Val Thr Ile Pro Asn Cys Se #r Lys Pro Arg Trp Glu    130               #   135               #   140Lys Phe Phe Met Val Thr Phe Ile Thr Ala Th #r Leu Trp Ile Ala Val145                 1 #50                 1 #55                 1 #60Phe Ser Tyr Ile Met Val Trp Leu Val Thr Il #e Ile Gly Tyr Thr Leu                165   #               170   #               175Gly Ile Pro Asp Val Ile Met Gly Ile Thr Ph #e Leu Ala Ala Gly Thr            180       #           185       #           190Ser Val Pro Asp Cys Met Ala Ser Leu Ile Va #l Ala Arg Gln Gly Leu        195           #       200           #       205Gly Asp Met Ala Val Ser Asn Thr Ile Gly Se #r Asn Val Phe Asp Ile    210               #   215               #   220Leu Val Gly Leu Gly Val Pro Trp Gly Leu Gl #n Thr Met Val Val Asn225                 2 #30                 2 #35                 2 #40Tyr Gly Ser Thr Val Lys Ile Asn Ser Arg Gl #y Leu Val Tyr Ser Val                245   #               250   #               255Val Leu Leu Leu Gly Ser Val Ala Leu Thr Va #l Leu Gly Ile His Leu            260       #           265       #           270Asn Lys Trp Arg Leu Asp Arg Lys Leu Gly Va #l Tyr Val Leu Val Leu        275           #       280           #       285Tyr Ala Ile Phe Leu Cys Phe Ser Ile Met Il #e Glu Phe Asn Val Phe    290               #   295               #   300Thr Phe Val Asn Leu Pro Met Cys Arg Glu As #p Asp 305                 3#10                 3 #15 <210> SEQ ID NO 5 <211> LENGTH: 1062<212> TYPE: DNA <213> ORGANISM: homo sapiens <400> SEQUENCE: 5atgcaagcct ttttcacagt caaacaaaag agcattgcaa acggtaaccc gg#tcaacagt     60gagctggagg ctgtgaagga gaagccacag tatggcaaga accccgtggt ga#tggtggac    120gagattatga gctccagccc tcccaagttc accttccctg aagcaggctt ac#gaatcatg    180atcaccaata agtttggacc caggacccga ctacggatgg ccagcaggat ca#tcattaat    240gagcggcaga gactgatcaa ctcggccaat ggtgtgagca gtaagccgct tc#aaaacggg    300aggcacgaga acattgagaa cgggaatgtt cctgtggaaa accccgaaga cc#ctcagcag    360aatcaggagc agcagccgcc gccacagcca ccaccgccag agccagagcc gg#tggaggct    420gacttcctgt cccccttctc cgtgccggag gccagagggg acaaggtcaa gt#gggtgttc    480acctggcccc tcatcttcct cctgtgcgtc accattccca actgcagcaa gc#cccgctgg    540gagaagttct tcatggtcac cttcatcacc gccacgctgt ggatcgctgt gt#tctcctac    600atcatggtgt ggctggtgac tattatcgga tacacacttg ggatcccgga tg#tcatcatg    660ggcattactt tcctggcagc agggacaagt gttccagact gcatggccag cc#taattgtg    720gcgagacaag gccttgggga catggcagtc tccaacacca taggaagcaa cg#tgtttgac    780atcctggtag gacttggtgt accgtggggc ctgcagacca tggttgttaa tt#atggatca    840acagtgaaga tcaacagccg ggggctggtc tattccgtgg tcctgttgct gg#gctctgtc    900gctctcaccg tcctcggcat ccacctaaac aagtggcgac tggaccggaa gc#tgggtgtc    960tacgtgctgg ttctctacgc catcttcttg tgcttctcca taatgataga gt#ttaacgtc   1020 tttaccttcg tcaacttgcc gatgtgccgg gaagacgatt ag    #                   #1062 <210> SEQ ID NO 6 <211> LENGTH: 353<212> TYPE: PRT <213> ORGANISM: homo sapiens <400> SEQUENCE: 6Met Gln Ala Phe Phe Thr Val Lys Gln Lys Se #r Ile Ala Asn Gly Asn 1               5   #                10   #                15Pro Val Asn Ser Glu Leu Glu Ala Val Lys Gl #u Lys Pro Gln Tyr Gly            20       #            25       #            30Lys Asn Pro Val Val Met Val Asp Glu Ile Me #t Ser Ser Ser Pro Pro        35           #        40           #        45Lys Phe Thr Phe Pro Glu Ala Gly Leu Arg Il #e Met Ile Thr Asn Lys    50               #    55               #    60Phe Gly Pro Arg Thr Arg Leu Arg Met Ala Se #r Arg Ile Ile Ile Asn65                   #70                   #75                   #80Glu Arg Gln Arg Leu Ile Asn Ser Ala Asn Gl #y Val Ser Ser Lys Pro                85   #                90   #                95Leu Gln Asn Gly Arg His Glu Asn Ile Glu As #n Gly Asn Val Pro Val            100       #           105       #           110Glu Asn Pro Glu Asp Pro Gln Gln Asn Gln Gl #u Gln Gln Pro Pro Pro        115           #       120           #       125Gln Pro Pro Pro Pro Glu Pro Glu Pro Val Gl #u Ala Asp Phe Leu Ser    130               #   135               #   140Pro Phe Ser Val Pro Glu Ala Arg Gly Asp Ly #s Val Lys Trp Val Phe145                 1 #50                 1 #55                 1 #60Thr Trp Pro Leu Ile Phe Leu Leu Cys Val Th #r Ile Pro Asn Cys Ser                165   #               170   #               175Lys Pro Arg Trp Glu Lys Phe Phe Met Val Th #r Phe Ile Thr Ala Thr            180       #           185       #           190Leu Trp Ile Ala Val Phe Ser Tyr Ile Met Va #l Trp Leu Val Thr Ile        195           #       200           #       205Ile Gly Tyr Thr Leu Gly Ile Pro Asp Val Il #e Met Gly Ile Thr Phe    210               #   215               #   220Leu Ala Ala Gly Thr Ser Val Pro Asp Cys Me #t Ala Ser Leu Ile Val225                 2 #30                 2 #35                 2 #40Ala Arg Gln Gly Leu Gly Asp Met Ala Val Se #r Asn Thr Ile Gly Ser                245   #               250   #               255Asn Val Phe Asp Ile Leu Val Gly Leu Gly Va #l Pro Trp Gly Leu Gln            260       #           265       #           270Thr Met Val Val Asn Tyr Gly Ser Thr Val Ly #s Ile Asn Ser Arg Gly        275           #       280           #       285Leu Val Tyr Ser Val Val Leu Leu Leu Gly Se #r Val Ala Leu Thr Val    290               #   295               #   300Leu Gly Ile His Leu Asn Lys Trp Arg Leu As #p Arg Lys Leu Gly Val305                 3 #10                 3 #15                 3 #20Tyr Val Leu Val Leu Tyr Ala Ile Phe Leu Cy #s Phe Ser Ile Met Ile                325   #               330   #               335Glu Phe Asn Val Phe Thr Phe Val Asn Leu Pr #o Met Cys Arg Glu Asp            340       #           345       #           350 Asp<210> SEQ ID NO 7 <211> LENGTH: 2366 <212> TYPE: DNA<213> ORGANISM: homo sapiens <400> SEQUENCE: 7ccgacctcgc cctcgggcca tgaggctttg gcccggagct cctcgcctct ga#gtcgcgca     60ccgcctgctc cagccccagc gccgctcggc cactgattgc actctggccg ct#gaagctcc    120ccatcctctc ccagagacgg cacccaggcg ctccgggatg gcgctccgcg gg#accctccg    180gccgctcaaa gttcgcagga ggcgagagat gctgccgcag caagtcggct tc#gtgtgcgc    240ggtgctggcc ctggtgtgct gtgcgtccgg cctcttcggc agcttggggc ac#aaaacagc    300ttctgctagc aaacgtgtcc tgccagacac atggagaaat agaaagttga tg#gccccagt    360gaatgggaca cagacagcca agaactgcac agatcctgcg attcacgagt tc#cccacaga    420tctgttctcc aataaggagc gacagcacgg agccgtcctg ctgcacatcc tt#ggtgctct    480gtatatgttc tatgccttgg ccatagtgtg cgatgacttc tttgttccgt ct#ctagagaa    540gatctgtgag agactccatc tgagcgaaga tgtggctgga gccaccttca tg#gctgcagg    600aagctcaacg ccagagctgt ttgcgtctgt tattggggtg ttcatcaccc ay#ggggacgt    660cggggtgggc accatcgtgg gctctgctgt gttcaacatc ctgtgcataa tt#ggagtgtg    720cggactgttt gctggccagg tggtccgtct gacgtggtgg gccgtgtgcc ga#gactccgt    780gtactacacc atctctgtca tcgtgctcat cgtgttcata tatgatgaac aa#attgtgtg    840gtgggaaggc ctggtgctca tcatcttgta tgtgttttat attctgatca tg#aagtacaa    900tgtgaagatg caagcctttt tcacagtcaa acaaaagagc attgcaaacg gt#aacccggt    960caacagtgag ctggaggctg tgaaggagaa gccacagtat ggcaagaacc cc#gtggtgat   1020ggtggacgag attatgagct ccagccctcc caagttcacc ttccctgaag ca#ggcttacg   1080aatcatgatc accaataagt ttggacccag gacccgacta cggatggcca gc#aggatcat   1140cattaatgag cggcagagac tgatcaactc ggccaatggt gtgagcagta ag#ccgcttca   1200aaacgggagg cacgagaaca ttgagaacgg gaatgttcct gtggaaaacc cc#gaagaccc   1260tcagcagaat caggagcagc agccgccgcc acagccacca ccgccagagc ca#gagccggt   1320ggaggctgac ttcctgtccc ccttctccgt gccggaggcc agaggggaca ag#gtcaagtg   1380ggtgttcacc tggcccctca tcttcctcct gtgcgtcacc attcccaact gc#agcaagcc   1440ccgctgggag aagttcttca tggtcacctt catcaccgcc acgctgtgga tc#gctgtgtt   1500ctcctacatc atggtgtggc tggtgactat tatcggatac acacttggga tc#ccggatgt   1560catcatgggc attactttcc tggcagcagg gacaagtgtt ccagactgca tg#gccagcct   1620aattgtggcg agacaaggcc ttggggacat ggcagtctcc aacaccatag ga#agcaacgt   1680gtttgacatc ctggtaggac ttggtgtacc gtggggcctg cagaccatgg tt#gttaatta   1740tggatcaaca gtgaagatca acagccgggg gctggtctat tccgtggtcc tg#ttgctggg   1800ctctgtcgct ctcaccgtcc tcggcatcca cctaaacaag tggcgactgg ac#cggaagct   1860gggtgtctac gtgctggttc tctacgccat cttcttgtgc ttctccataa tg#atagagtt   1920taacgtcttt accttcgtca acttgccgat gtgccgggaa gacgattagc gc#tgagtcgc   1980ggcccctggg agctgatctg gacaccctgt gacactggcg tcctcctctc cc#ctccttcc   2040cccaccacag gtctctcctg cataggcagc cactgtccgt tctttcacac ac#tggaagga   2100agagccatcg tggtctttgt ctggccacag gccaggctgc tgggcatcct cc#tcctcctt   2160ggagttccac ccctgcaagg ctggatttgg gggccattat ctgagcagct tc#aaagaccc   2220ctgagctgcc aaccacggag atgtgccaag catctcatct ctcctgcaca ct#ttagtcag   2280aaggacttct gcatgcagtt tgtctttctg ttctgcaggc agcttcagaa tt#gaggtcat   2340 ttgtgagcac aagatctcat agggca          #                   #            2366

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence encoding an amino acid sequence drawn from the groupconsisting of SEQ ID NOS: 2, 4, and
 6. 2. An isolated nucleic acidmolecule comprising a nucleotide sequence that: (a) encodes the aminoacid sequence shown in SEQ ID NO: 2; and (b) hybridizes to thenucleotide sequence of SEQ ID NO:1 or the complement thereof.
 3. Anisolated nucleic acid molecule comprising a nucleotide sequence encodingthe amino acid sequence shown in SEQ ID NO:2.
 4. An isolated nucleicacid molecule comprising a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO:4.
 5. An isolated nucleic acid moleculecomprising a nucleotide sequence encoding the amino acid sequence shownin SEQ ID NO:6.
 6. A recombinant expression vector comprising anisolated nucleic acid molecule of claim
 1. 7. A host cell comprising therecombinant expression vector of claim 6.